The present invention relates to a process for efficiently producing dipeptides or dipeptide derivatives from amino acids or amino acid derivatives.
Chemical synthesis methods (liquid phase method and solid phase method), enzymatic synthesis methods and biological synthesis methods utilizing recombinant DNA techniques are known as the methods for large-scale peptide synthesis. Currently, the enzymatic synthesis methods and biological synthesis methods are employed for the synthesis of long-chain peptides longer than 50 residues, and the chemical synthesis methods and enzymatic synthesis methods are mainly employed for the synthesis of dipeptides.
In the synthesis of dipeptides by the chemical synthesis methods, operations such as introduction and removal of protective groups for functional groups are necessary, and racemates are also formed. The chemical synthesis methods are thus considered to be disadvantageous in respect of cost and efficiency. They are unfavorable also from the viewpoint of environmental hygiene because of the use of large amounts of organic solvents and the like.
As to the synthesis of dipeptides by the enzymatic methods, the following methods are known: a method utilizing reverse reaction of protease (J. Biol. Chem., 119, 707-720 (1937)); methods utilizing thermostable aminoacyl t-RNA synthetase (Japanese Published Unexamined Patent Application No. 146539/83, Japanese Published Unexamined Patent Application No. 209991/83, Japanese Published Unexamined Patent Application No. 209992/83 and Japanese Published Unexamined Patent Application No. 106298/84); and methods utilizing non-ribosomal peptide synthetase (hereinafter referred to as NRPS) (Chem. Biol., 7, 373-384 (2000), FEBS Lett., 498, 42-45 (2001), U.S. Pat. No. 5,795,738 and U.S. Pat. No. 5,652,116).
However, the method utilizing reverse reaction of protease requires introduction and removal of protective groups for functional groups of amino acids used as substrates, which causes difficulties in raising the efficiency of peptide-forming reaction and in preventing peptidolytic reaction. The methods utilizing thermostable aminoacyl t-RNA synthetase have the defects that the expression of the enzyme and the prevention of side reactions forming by-products other than the desired products are difficult. The methods utilizing NRPS are inefficient because NRPS requires adenosine-5′-triphosphate (ATP) for reaction and it is necessary to add a large amount of ATP to the reaction system.
A group of peptide synthetases such as γ-glutamylcysteine synthetase, glutathione synthetase, D-alanyl-D-alanine (D-Ala-D-Ala) ligase and poly-γ-glutamate synthetase are also known as proteins having dipeptide-synthesizing activity. However, most of these enzymes utilize D-amino acids as substrates or catalyze peptide bond formation at the γ-carboxyl group. Because of such properties, they can not be used for the synthesis of dipeptides by peptide bond formation at the α-carboxyl group of L-amino acid.
The only known example of an enzyme capable of dipeptide synthesis by the activity to form a peptide bond at the α-carboxyl group of L-amino acid is bacilysin (dipeptide antibiotic derived from a microorganism belonging to the genus Bacillus) synthetase. Bacilysin synthetase is known to have the activity to synthesize bacilysin [L-alanyl-L-anticapsin (L-Ala-L-anticapsin)] and L-alanyl-L-alanine (L-Ala-L-Ala), but there is no information about its activity to synthesize other dipeptides (J. Ind. Microbiol., 2, 201-208 (1987) and Enzyme. Microbial. Technol., 29, 400-406 (2001)).
As for the bacilysin biosynthetase genes in Bacillus subtilis 168 whose entire genome information has been clarified (Nature, 390, 249-256 (1997)), it is known that the productivity of bacilysin is increased by amplification of bacilysin operons containing ORFs ywfA-F (WO00/03009 pamphlet). However, it is not known whether an ORF encoding a protein having the activity to ligate two or more amino acids by peptide bond is contained in these ORFs, and if contained, which ORF encodes the protein.
It is reported that a protein bearing no similarity to NRPS (albC gene product) is responsible for the synthesis of the cyclo(L-phenylalanyl-L-leucine) structure in Streptomyces noursei ATCC 11455 known as a strain producing the antibiotic albonoursin and that albonoursin was detected when cyclo dipeptide oxidase was made to act on the culture liquor of Escherichia coli and Streptomyces lividans into which the albC gene was introduced (Chemistry & Biol., 9, 1355-1364 (2002)). However, there is no report that the albC gene product forms a straight-chain dipeptide.
As to the method of supplying ATP, which is an energy source in various enzyme reactions, regeneration of ATP from ADP utilizing the glycolytic pathway and regeneration of ATP from ADP utilizing polyphosphate kinase and polyphosphoric acid (Agric. Biol. Chem., 52, 1471-1477 (1988), Biotech. Appl. Biochem., 10, 107-117 (1988) and Biotech. Appl. Biochem., 15, 125-133 (1992)) are known. The system of regenerating ATP from ADP utilizing the glycolytic pathway is present in all the microorganisms having the glycolytic pathway. Polyphosphate kinase capable of regenerating ATP from ADP utilizing polyphosphoric acid is known to be widely present in bacteria (Agric. Biol. Chem., 52(6), 1471-1477 (1988), Biotech. Appl. Biochem., 10, 107-117 (1988), Biotech. Appl. Biochem., 15, 125-133 (1992) and J. Biol. Chem., 267, 22556-22561 (1992)), yeast (J. Biol. Chem., 234, 2595-2604 (1959) and Arch. Biochem. Biophys., 83, 259-267 (1959)), plants (Biochem. J., 124, 407-417 (1971)) and animals (Biochem. J., 75, 417-428 (1960)).
However, it is not known that dipeptides can be efficiently produced by combining dipeptide-forming reaction requiring ATP and ATP-regenerating reaction utilizing polyphosphate kinase and polyphosphoric acid.
An object of the present invention is to provide a process for efficiently producing dipeptides or dipeptide derivatives.